1. Field of the Invention
The present invention relates to a binding assay for the detection of Mycobacteria in fluids. In a preferred embodiment, the invention relates to a method for the diagnosis for tuberculosis in humans.
2. Description of the Prior Art
Mycobacteria cause a wide variety of infections both in humans and non-human animals. For example, M. tuberculosis is the causative organism of human tuberculosis; it was isolated and identified in 1882. M. leprae is the cause of Hansen's disease and M. lepraemurium is the cause of a leprosy-like disease in rats. M. bovis is the cause of cattle infections, and it, like M. tuberculosis also causes infections in man. M. avium is strongly pathogenic for fowl, yet not pathogenic for man. Other tubercle bacilli of the Mycobaterium genus are the so-called cold-blooded animal type and the saprophytic types. The latter two are not pathogenic for man.
M. tuberculosis is an almost exclusive parasite of man. It is responsible for over 90% of all cases of tuberculosis, whereas the bovine type produces tuberculosis in man through ingestion of infected beef, or milk from an infected cow. Although infection from M. bovis, generally manifested as tuberculosis of the bones or lymphatic system, has been largely eliminated as a source of human infection in the United States as a result of Government inspection of meats, infection due to M. tuberculosis is still a major world health problem. According to the World Health Organization, there were 15 to 20 million infectious cases of tuberculosis in the world in 1967. The report for 1967 also stated that two to three million deaths due to tuberculosis occur each year with 80% of the deaths in developing countries. (Pelczar, Jr. and Reid, "Microbiology," Third Edition, McGraw-Hill (1972), p.78). In 1976, more than 32,000 new cases were reported in the United States alone. Eradication of tuberculosis in man and similar infections in non-human animals is therefore still of utmost significance in the United States and the rest of the world.
Detection of actively growing Mycobacteria in human and non-human animals has been carried out in the prior art by classical staining and culture methodology. Mycobacteria are difficult to stain with the usual microbiological dyes, but they stain readily by the Ziehl-Neelsen technique (initial staining with basic fuchsin washed with acid and alcohol). Probably because of the high fat content of the organisms, they are not decolorized by the acid-alcohol and therefore have been termed acid-fast organisms. The requirement for isolation and culture of Mycobacteria from relatively inaccessible organs coupled with the slow multiplication rate of the tubercle bacillus, presents a problem for rapid diagonosis. In fact, since tuberculosis is a chronic bacterial disease, advancing slowly, the primary infection may go unnoticed until a chance x-ray reveals lung lesions.
The demonstration of tubercle bacilli in body discharges--sputum, gastric contents, spinal fluid, urine, etc.--is the final proof in corroboration of clinical diagnosis. Laboratory methods include microscope examination of stained samples for the presence of bacilli, planting the suspected material on suitable culture media, and animal innoculation with concentrated sputum or other material in which the organisms may be found.
Another diagnostic method is the use of the tuberculin test. The test is performed by injecting intracutaneously small amounts of tuberculin, a purified protein derivative from cultured tuberculosis bacilli. A positive test is indicated by an inflammatory reaction at the site of injection within 48 hours. A positive tuberculin test, however, is not necessarily an indication of an existing infection in adults for it may simply mean that they once harbored the bacilli or some non-pathogenic Mycobacterium or an atypical M. tuberculosis. In children, a positive test is usually an indication for treatment. X-ray examination is generally employed to corroborate pulmonary infection. However, at present only identification of Mycobacteria in cultures of clinical specimens can be considered proof of active disease. Alternative and somewhat more efficient methods for rapid detection of growing Mycobacteria, especially for the rapid diagnosis for tuberculosis, have been proposed in the prior art. Thus, Odham et al (J. Clin. Invest. 63:813-819 (1979)), have demonstrated the presence of tuberculostearic acid in sputum from patients with pulmonary tuberculosis by selective ion monitoring.
Straus and Yalow (Clinical Research, Volume 25, No. 3, April 1977, A384) describe a radioimmunoassay (RIA) procedure for the detection of tuberculin purified protein derivative (PPD) shed into culture media by growing Mycobacterium tuberculosis. In this procedure, antisera were raised in guinea pigs by repeated subcutaneous injection of PPD (a readily available commercial product). PPD was iodinated with .sup.125 I and analysis of the radiolabeled PPD by Sepharose 6B column chromatography yielded a peak of radioactivity in the void volume (VVP) which was used as a tracer in RIA. Separation of the antibody-bound and free tracer was achieved by precipitation with rabbit anti-guinea pig .gamma.-globulin. This radioimmunoassay is not useful, however, for the detection of M. tuberculosis or any other Mycobacterial species which are actively growing in an animal host such as a human or non-human infected animal. The Straus and Yalow assay (as described in the last two lines of the Abstract) was not sensitive enough to be employed for the rapid and direct identification of M. tuberculosis in biological fluids. The void volume peak (VVP) is generally too unstable to provide suitably labeled antigens and has the further disadvantage that precipitation with anti-guinea pig globulin is necessary to separate free from bound labeled antigens, since the VVP does not absorb on to charcoal.
A need, therefore, exists for a highly sensitive, rapid and efficient method for the detection of actively growing Mycobacteria in human and nonhuman animals. More particularly, a need exists for a rapid diagnostic method for the many different forms of tuberculosis in humans.